Diabetes mellitus, a chronic group of disorders characterized by hyperglycemia, remains a major cause of premature disability and mortality in United States. Patients with type I diabetes have little or no insulin secretory capacity, and the primary focus for their treatment is to replace insulin secretion by the administration of exogenous insulin. However, optimal glycemic control is often difficult to achieve. The broad, long-term objective of this proposal is to engineer a system in which insulin secretion in response to food intake will ensure tight control of blood glucose levels. As a first step in devising gene therapy strategies for treatment of diabetes, we have chosen to utilize the gastrin-secreting G cells of the stomach to produce surrogate a cells. Our central hypothesis is that human insulin, produced by G cells under the regulation of the gastrin gene promoter, would be secreted hi response to meal-associated stimuli. In order to explore this hypothesis, we have demonstrated that the gastrin promoter can target expression of insulin to G cells and, consequently, we are generating transgenic knock-in mice G-InsKI, in which the coding sequence of human insulin is knocked into the mouse gastrin gene. Specifically, insulin produced by and stored in the gastric G cells of G-InsKI mice is envisaged to be released in response to either hormonal regulators, including bombesin/GRP, or by luminal regulators, including aromatic amino acids, Ca 2+ and tastants. These stimuli act directly via specific G protein-coupled receptors on the surface of G cells. The elucidation of the signaling pathways involved could provide novel therapeutic approaches to regulate the production and secretion of insulin from the G cells. Our central hypothesis will be tested by pursuing the following specific aims: 1: Characterize G-InsKI mice and assess regulated release of insulin from the gastric G cells in G-InsKI mice. 2: Identify the signal transduction pathways that control the secretion of insulin from gastrointestinal endocrine cells using the hormone secreting enteroendocrine STC-1 cell line as a model of G cells.